The present invention relates to the method of analyzing a primary structure of protein or peptide.
Conventionally, in order to determine an amino acid sequence of protein or peptide from a carboxy-terminal or C-terminal, as shown in FIG. 2, carboxypeptidase is applied to protein or peptide. Then, a digested solution is sampled time-sequentially, and the sampled digested solution is analyzed by an amino acid analyzer to effect quantitative measurement of free amino acids. Such method is described in "Biochemical Experiment Text, Vol. 1, Chemistry of Protein II, pp 203-211, 1976, edited by Japanese Biochemistry Society".
There has been reported another method that the digested solution is treated by a mass spectrometer to measure mass of protein or peptide which partially lost amino acids from the C-terminal. Such method is disclosed in "A. Tsugita, R. van den Broek, M. Pyzybylski, FEBS. Lett. 137, 19(1982)".
Further, there has been reported a still another method in "D. H. Hawke, H-. W. Lahm, J. E. Shively, C. W. Todd, Anal. Biochem. 166, 298(1987)". As shown in FIG. 3, the C-terminal is activated by acetic acid anhydride and is then coupled to trimethylsililisothiocyanate (TMS-ITC). Thereafter, the protein or peptide is cut by hydrochloric acid. These sequential treatments are repeatedly undertaken to perform the amino acid sequence analysis.
The conventional method utilizing carboxypeptidase has drawbacks that the substrate specificity and the activity of the carboxypeptidase vary dependently on a C-terminal amino acid or an adjacent amino acid; that the reagent contains other contaminant enzymes which hinder a precise analysis; and that an external amino acid is introduced by self-dissociation of the enzyme to thereby hinder sensitivity of the amino acid analysis. The other conventional method utilizing TMS-ITC has drawbacks that the processing is complicated since three kinds of reagents are applied sequentially and that repititive yield rate is rather small, thereby not being practical.